The obesity epidemic is threatening world health, largely because of its associated diseases, type 2 diabetes mellitus and atherosclerosis. Although the mechanisms underlying obesity-associated diseases are unclear, the lipotoxicity hypothesis has emerged as a plausible explanation. This hypothesis states that the deposition of excess lipids in tissues other than white adipose tissue over time leads to tissue dysfunction. For example, lipid deposition in skeletal muscle is associated with insulin resistance, in pancreatic beta cells with defective insulin secretion, and in heart muscle with cardiomyopathy. Despite these strong associations from studies in animals and humans, the lipotoxicity hypothesis remains unproven, and its underlying mechanisms remain unclear. Whether triglycerides themselves or precursors of triglyceride synthesis (e.g., diacylglycerol and fatty acyl CoAs) are toxic to nonadipose cells is unclear. We propose to test the lipotoxicity hypothesis by directly modulating triglyceride synthesis in specific tissues of mice. Triglyceride synthesis is catalyzed by acyl CoA:diacylglycerol (DGAT) enzymes. Aim 1 describes the generation and analysis of mice that overexpress DGAT1 in skeletal muscle, pancreatic beta cells, and cardiac muscle. Aim 2 describes the generation and analysis of mice that lack DGAT1 in skeletal muscle, beta cells, and white adipose tissue. We will use these mouse models to determine whether modulating triglyceride synthesis influences tissue lipotoxicity and to explore the mechanisms, including alterations in tissue lipids, gene expression, and signaling pathways, that contribute to lipotoxicity.